Methods, compositions, and kits for the selective activation of protoxins through combinatorial targeting
The present invention provides methods and compositions for treating various diseases through selective killing of targeted cells using a combinatorial targeting approach. The invention features protoxin fusion proteins containing a cell targeting moiety and, a modifiable activation moiety which is activated by an activation moiety not naturally operably found in, on, or in the vicinity of a target cell. These methods also include the combinatorial use of two or more therapeutic agents, at minimum comprising a protoxin and a protoxin activator, to target and destroy a specific cell population.
1. A composition comprising;
(i) a protoxin fusion protein comprising a first non-native cell-targeting moiety, a selectively modifiable activation domain and a toxin domain; and a
(ii) protoxin activator fusion protein comprising a second non-native cell-targeting moiety and a modification domain;
wherein:
said first cell-targeting moiety of said protoxin fusion protein and said second cell-targeting moiety of said protoxin activator fusion protein each recognize and bind a common target cell;
said modification domain comprises protease or phosphatase enzymatic activity exogenous to said target cell;
said selectively modifiable activation domain comprises a substrate for said modification domain; and
modification of said selectively modifiable activation domain by said modification domain results in activation of said toxin domain.
2. The composition of claim 1 , wherein said enzymatic activity is protease activity.
3. The composition of claim 1 , wherein said modification domain is a phosphatase and said modifiable activation domain comprises phosphorylation of a protease cleavage site.
4. The composition of claim 1 , wherein at least one non-native cell-targeting moiety is an artificially diversified binding protein.
5. The composition of claim 1 , wherein said protoxin is an activatable toxin.
6. The composition of claim 5 , wherein said activatable toxin is selected from the group consisting of an activatable pore forming toxin or an activatable enzymatic toxin.
7. The composition of claim 1 , wherein said toxin domain is selected from a group consisting of an AB toxin, a cyotoxic necrotizing factor toxin, a dermonecrotic toxin, and an activatable ADP-ribosylating toxin.
8. The composition of claim 1 , wherein said toxin domain is selected from a group consisting of aerolysin, Vibrio cholerae exotoxin, Pseudomonas exotoxin and diphtheria toxin.
9. The composition of claim 1 , wherein said protoxin activator fusion protein further comprises a natively activatable domain wherein said modification domain is inactive prior to activation of said natively activatable domain and, when active, is non-toxic to a target cell.
10. The composition of claim 1 , wherein said modification domain is a protease domain.
11. The composition of claim 10 , wherein said protease domain is the catalytic domain of a non-human protease.
12. The composition of claim 11 , wherein said non-human protease is a viral protease.
13. The composition of claim 1 , wherein said non-native cell-targeting moiety recognizes a cancer cell.
14. The composition of claim 1 , wherein at least one non-native cell-targeting moiety is an antibody or antibody fragment.
15. The composition of claim 1 , wherein both of said cell-targeting moieties is an antibody or antibody fragment.
16. The composition of claim 10 , wherein said protease domain is the catalytic domain of an exogenous human protease.
17. A composition comprising:
(i) a protoxin fusion protein comprising a first non-native cell-targeting moiety, a selectively modifiable activation domain and a toxin domain; and a
(ii) protoxin activator fusion protein comprising a second non-native cell-targeting moiety and a modification domain;
wherein:
said first cell-targeting moiety of said protoxin fusion protein and said second cell-targeting moiety of said protoxin activator fusion protein each recognize and bind a common target cell;
said modification domain comprises enzymatic activity exogenous to said target cell;
said selectively modifiable activation domain comprises a substrate for said modification domain; and
modification of said selectively modifiable activation domain by said modification domain results in proteolytic cleavage and activation of said toxin domain.